The present invention is generally in the field of regulatory proteins which exert their effects by intracellular signaling processes which are mediated by regulatory elements (domains or motifs) contained within the intracellular domains of these proteins. More specifically, the present invention concerns new modulators being proteins, peptides, antibodies or analogs or fragments of any thereof, and organic compounds which are capable of interacting with, or binding to the newly discovered xe2x80x98death domainxe2x80x99 motif present in a wide range of related and unrelated proteins, for example, receptors of the TNF/NGF family such as p55 TNF-R, FAS-R, NGF-R, a related protein MORT-1, proteins known as TRADD and RIP and the unrelated protein ankyrin 1. These new modulators are capable of modulating or regulating the activity of the proteins which contain the xe2x80x98death domainxe2x80x99 motif.
There is a very large group of regulatory proteins which exert their regulatory effects on cells by way of intracellular signaling processes, mediated by regulatory portions or motifs contained within these proteins. Members of this group of proteins include, receptors belonging to the TLF/NGF family of receptors, such as, for example, the p55 and p75 TNF receptors (p55 and p75 TNF-Rs), the NGF receptor (NGF-R) and the Fas/APO1 protein (also called the FAS-ligand receptor or FAS-R, and hereinafter will be called FAS-R); these receptors being characterized by having an extracellular ligand-binding domain, a transmembrane domain and an intracellular (IC) domain, which intracellular domain, or portions thereof, is involved in the mediation of the intracellular signaling events initiated by the binding of the ligand to the extracellular domain. Other members of this group include various intracellular proteins, for example, the cytoskeleton-associated structural proteins, the ankyrins, which have a regulatory domain that is possibly involved in the ability of these proteins to associate with or bind to other cytoskeletal proteins, e.g. spectrin, or to other transmembrane proteins. Yet another member of this group is the recently identified MORT1 protein (also called HF1, see co-pending IL 112002 and EL 112692), which is capable of binding specifically to the intracellular domain of the FAS-R, and which is also capable of self-association and of mediating, in a ligand-independent manner, cytotoxic effects on cells. In MORT-1, a regulatory domain was also identified (see IL 112692).
Tumor Necrosis Factor (TNF-xcex1) and Lymphotoxin (TNF-xcex2) (hereinafter, TNF, refers to both TNF-xcex1 and TNF-xcex2) are multifunctional pro-inflammatory cytokines formed mainly by mononuclear phagocytes, which have many effects on cells (Wallach, D. (1986) in: Interferon 7 (Ion Gresser, ed.), pp. 83-122, Academic Press, London; and Beutler and Cerami (1987)). Both TNF-xcex1 and TNF-xcex2 initiate their effects by binding to specific cell surface receptors. Some of the effects are likely to be beneficial to the organism: they may destroy, for example tumor cells or virus infected cells and augment antibacterial activities of granulocytes. In this way, TNF contributes to the defense of the organism against tumors and infectious agents and contributes to the recovery from injury. Thus, TNF can be used as an anti-tumor agent in which application it binds to its receptors on the surface of tumor cells and thereby initiates the events leading to the death of the tumor cells. TNF can also be used as an anti-infectious agent.
However, both TNF-xcex1 and TNF-xcex2 also have deleterious effects. There is evidence that over-production of TNF-xcex1 can play a major pathogenic role in several diseases. Thus, effects of TNF-xcex1, primarily on the vasculature, are now known to be a major cause for symptoms of septic shock (Tracey et al., 1986). In some diseases, TNF may cause excessive loss of weight (cachexia) by suppressing activities of adipocytes and by causing anorexia, and TNF-xcex1 was thus called cachetin. It was also described as a mediator of the damage to tissues in rheumatic diseases (Beutler and Cerami, 1987) and as a major mediator of the damage observed in graft-versus-host reactions (Piquet et al., 1987). In addition, TNF is known to be involved in the process of inflammation and in many other diseases.
Two distinct, independently expressed, receptors, the p55 and p75 TNF-Rs, which bind both TNF-xcex1 and TNF-xcex2 specifically, initiate and/or mediate the above noted biological effects of TNF. These two receptors have structurally dissimilar intracellular domains suggesting that they signal differently (See Hohmann et al., 1989; Engelmann et al., 1990; Brockhaus et al., 1990; Loetscher et al., 1990; Schall et al., 1990; Nophar et al., 1990; Smith et al., 1990; and Heller et al., 1990). However, the cellular mechanisms, for example, the various proteins and possibly other factors, which are involved in the intracellular signaling of the p55 an p75 TNF-Rs have yet to be elucidated (In IL 109632 there are described for the first time, new proteins capable of binding to the intracellular domains of p55 and p75 TNF-Rs, these intracellular domains being called, respectively, p75IC and p55 IC). It is this intracellular signaling, which occurs usually after the binding of the ligand, i.e. TNF (xcex1 or xcex2), to the receptor, that is responsible for the commencement of the cascade of reactions that ultimately result in the observed response of the cell to TNF.
As regards the above mentioned cytocidal effect of TNF, in most cells studied so far, this effect is triggered mainly by the p55 TNF-R. Antibodies against the extracellular domain (ligand binding domain) of the p55 TNF-R can themselves trigger the cytocidal effect (see EP 412486) which correlates with the effectivity of receptor cross-linking by the antibodies, believed to be the first step in the generation of the intracellular signaling process. Further, mutational studies (Brakebusch et al., 1992; Tartaglia et al., 1993) have shown that the biological function of the p55 TNF-R depends on the integrity of its intracellular domain, and accordingly it has been suggested that the initiation of intracellular signaling leading to the cytocidal effect of TNF occurs as a consequence of the association of two or more intracellular domains of the p55 TNF-R. Moreover, TNF (xcex1 and xcex2) occurs as a homotrimer and as such has been suggested to induce intracellular signaling via the p55 TNF-R by way of its ability to bind to and to cross-link the receptor molecules, i.e. cause receptor aggregation. In co-pending IL 109632 and IL 111125, there is described how the p55IC and p55DD can self-associate and induce, in a ligand-independent fashion, TNF-associated effects in cells.
Another member of the TNF/NGF superfamily of receptors is the FAS receptor (FAS-R) which has also been called the Fas antigen, a cell-surface protein expressed in various tissues and sharing homology with a number of cell-surface receptors including TNF-R and NGF-R. The FAS-R mediates cell death in the form of apoptosis (Itoh et al., 1991), and appears to serve as a negative selector of autoreactive T cells, i.e. during maturation of T cells, FAS-R mediates the apoptopic death of T cells recognizing self-antigens. It has also been found that mutations in the FAS-R gene (lpr) cause a lymphoproliferation disorder in mice that resembles the human autoimmune disease systemic lupus erythematosus (SLE) (Watanabe-Fukunaga et al., 1992). The ligand for the FAS-R appears to be a cell-surface associated molecule carried by, amongst others, killer T cells (or cytotoxic T lymphocytesxe2x80x94CTLs), and hence when such CTLs contact cells carrying FAS-R, they are capable of inducing apoptopic cell death of the FAS-R-carrying cells. Further, a monoclonal antibody has been prepared that is specific for FAS-R, this monoclonal antibody being capable of inducing apoptopic cell death in cells carrying FAS-R, including mouse cells transformed by cDNA encoding human FAS-R (Itoh et al., 1991).
It has also been found that various other normal cells, besides T lymphocytes, express the FAS-R on their surface and can be killed by the triggering of this receptor. Uncontrolled induction of such a killing process is suspected to contribute to tissue damage in certain diseases, for example, the destruction of liver cells in acute hepatitis. Accordingly, finding ways to restrain the cytotoxic activity of FAS-R may have therapeutic potential.
Conversely, since it has also been found that certain malignant cells and HIV-infected cells carry the FAS-R on their surface, antibodies against FAS-R, or the FAS-R ligand, may be used to trigger the FAS-R mediated cytotoxic effects in these and thereby provide a means for combating such malignant cells or HFV-infected cells (see Itoh et al., 1991). Finding yet other ways for enhancing the cytotoxic activity of FAS-R may therefore also have therapeutic potential.
In co-pending IL 109632, IL 111125 and IL 112002 there is described that the intracellular domain of FAS-R, the so-called FAS-IC, is capable of self-association and contains within this intracellular domain a region called the xe2x80x98death domainxe2x80x99 (DD) which is primarily responsible for the self-association of the FAS-IC. This xe2x80x98death domainxe2x80x99 shares sequence homology with the p55 TNF-R, xe2x80x98death domainxe2x80x99 (p55DD).
It has been a long felt need to provide a way for modulating the cellular response to TNF (xcex1 or xcex2) and FAS-R ligand, for example, in pathological situations as mentioned above, where TNF or FAS-R ligand is over-expressed it is desirable to inhibit the TNF- or FAS-R ligand-induced cytocidal effects, while in other situations, e,g. wound healing applications, it is desirable to enhance the TNF effect, or in the case of FAS-F, in tumor cells or HIV-infected cells it is desirable to enhance the FAS-R mediated effect.
A number of approaches have been made by the present inventors (see for example, European Application Nos. EP 186833, EP 308378, EP 398327 and EP 412486) to regulate the deleterious effects of TNF by inhibiting the binding of TNF to its receptors using anti-TNF antibodies or by using soluble TNF receptors (being essentially the soluble extracellular domains of the receptors) to compete with the binding of TNF to the cell surface-bound TNF-Rs. Further, on the basis that TNF-binding to its receptors is required for the TNF-induced cellular effects, approaches by the present inventors (see for example IL 101769 and its corresponding EP 568925) have been made to modulate the TNF effect by modulating the activity of the TNF-Rs. Briefly, EP 568925 (IL 101769) relates to a method of modulating signal transduction and/or cleavage in TNF-Rs whereby peptides or other molecules may interact either with the receptor itself or with effector proteins interacting with the receptor, thus modulating the normal functioning of the TNF-Rs. In EP 568925 there is described the construction and characterization of various mutant p55 TNF-Rs, having mutations in the extracellular, transmembranal, and intracellular domains of the p55 TNF-R. In this way regions within the above domains of the p55 TNF-R were identified as being essential to the functioning of the receptor, i.e. the binding of the lizand (TNF) and the subsequent signal transduction and intracellular signaling which ultimately results in the observed TNF-effect on the cells. Further, there is also described a number of approaches to isolate and identify proteins, peptides or other factors which are capable of binding to the various regions in the above domains of the TNF-R, which proteins, peptides and other factors may be involved in regulating or modulating the activity of the TNF-R. A number of approaches for isolating and cloning the DNA sequences encoding such proteins and peptides; for constructing expression vectors for the production of these proteins and peptides; and for the preparation of antibodies or fragments thereof which interact with the TNF-R or with the above proteins and peptides that bind various regions of the TNF-R, are also set forth in EP 568925. However, no description is made in EP 568925 of the actual proteins and peptides which bind to the intracellular domains of the TNF-Rs (e.g. p55 TNF-R), nor is any description made of the yeast two-hybrid approach to isolate and identify such proteins or peptides which bind to the intracellular domains of TNF-Rs. Similarly, heretofore there has been no disclosure of proteins or peptides capable of binding the intracellular domain of FAS-R.
Thus, when it is desired to inhibit the effect of TNF, or the FAS-R ligand, it would be desirable to decrease the amount or the activity of TNF-Rs or FAS-R at the cell surface, while an increase in the amount or the activity of TNF-Rs or FAS-R would be desired when an enhanced TNF or FAS-R ligand effect is sought. To this end the promoters of both the p55 TNF-R and the p75 TNF-R have been sequenced, analyzed and a number of key sequence motifs have been found that are specific to various transcription regulating factors, and as such the expression of these TNF-Rs can be controlled at their promoter level, i.e. inhibition of transcription from the promoters for a decrease in the number of receptors, and an enhancement of transcription from the promoters for an increase in the number of receptors (see IL 104355 and EL 109633). Corresponding studies concerning the control of FAS-R at the level of the promoter of the FAS-R gene have yet to be reported.
Further, it should also be mentioned that, while it is known that the tumor necrosis factor (TNF) receptors, and the structurally-related receptor FAS-R, trigger in cells, upon stimulation by leukocyte-produced ligands, destructive activities that lead to their own demise, the mechanisms of this triggering are still little understood. Mutational studies indicate that in FAS-R and the p55 TNF receptor (p55-R) signaling for cytotoxicity involve distinct regions within their intracellular domains (Brakebusch et al., 1992; Tartaglia et al., 1993; Itoh and Nagata, 1993). These regions (the xe2x80x98death domainsxe2x80x99) have sequence similarity. The xe2x80x98death domainsxe2x80x99 of both FAS-R and p55-R tend to self-associate. Their self-association apparently promotes that receptor aggregation which is necessary for initiation of signaling (see IL 109632, IL 111125 and IL 1 12002, as well as Song et al., 1994; Wallach et al., 1994; Boldin et al., 1995) and at high levels of receptor expression can result in triggering of ligand-independent signaling (IL 109632, IL 111125 and Boldin et al., 1995).
The ankyrins constitute a family of proteins that control interactions between integral membrane components and cytoskeletal elements and are found in a wide range of tissues such as brain tissue and in erythrocytes, the erythrocyte ankyrin being the best characterized. The ankyrins are intracellular proteins associated with the cytoskeletal elements of the cell and have three domains: an upper domain involved in binding to the intracellular domains of transmembrane proteins, this upper domain containing the well-known repeats, the so-called ankyrin repeats; a middle domain which is involved in binding to spectrin, i.e. the binding of spectrin to transmembrane proteins via the ankyrins; and a C-terminal or lower (or third) domain, which is the regulatory domain that is capable of being phosphorylated, this domain regulating the activity of the other two domains when phosphorylated or dephosphorylated. This latter regulatory domain also has three parts: a middle part that can be deleted by alternative splicing naturally, and hence some ankyrins have this Part and others don""t; and two other parts, less-well characterized (for a review on the ankyrins, see Lux et al., 1990 and Lambert and Bennett, 1993).
It should be noted however, as is set forth hereinbelow, that in accordance with the present invention, it has been discovered that the upper part of the above noted regulatory (C-terminal) domain of ankyrin contains a so-called xe2x80x98death domainxe2x80x99 motif, which may function to mediate the binding of proteins together (activity of the first two ankyrin domains), or may function conformationally to regulate the arkyrin protein.
The NGF-R is a low affinity NGF receptor which is not well characterized. The NGF-R is considered to be involved in growth regulation, such as its possible involvement in signaling intracellularly for NGF-induced effects. However, a recent publication discloses that overexpression of NGF-R in the absence of NGF can cause cell death. Thus, NGF-R appears to have a regulatory role in cell viability (see Rabizadeh et al. 1993).
It should be noted however, as is set forth hereinbelow, that in accordance with the present invention, it has been discovered that the NGF-R contains a xe2x80x98death domainxe2x80x99 motif in its intracellular domain, which may be involved in the mediation of the intracellular events associated with the regulatory role played by NGF-R with regards to cell viability.
MORT-1 is a recently discovered protein that binds to the intracellular domain of FAS-R, is capable of self-association and can activate cell cytotoxicity on its own. Hence, MORT1 is also a regulatory protein involved in intracellular signaling processes. It was also discovered that MORT-1 has a xe2x80x98death domainxe2x80x99 motif that is associated with its observed biological activity (see co-pending IL 112002 and IL 112692).
Two further intracellular proteins, RIP (Stanger et al., 1995) and TRADD (Hsu et al., 1995), that bind to the intracellular domains of p55 TNF-R or FAS-R and apparently take part in the induction of their cytocidal effect, have recently been cloned. All three proteins, MORT-1, RIP and TRADD, were found to contain the sequence motif shared between the xe2x80x98death domainsxe2x80x99 of the intracellular domains of p55-TNF-R and FAS-R. As in the receptors, the xe2x80x98death domainxe2x80x99 motifs (DD) in the three intraceuular proteins seem to be sites of protein-protein interaction. The three proteins interact with the p55-TNF-R and FAS-R intracellular domains by the binding of their DDs to those in the receptors, and in both TRADD and RIP (though not in MORT-1) the DDs self-associate. It has now been found that MORT-1 and TRADD bind differentially to FAS-R and p55 TNF-R and also bind to each other. Moreover, both bind effectively to RIP.
Interference of the interaction between the above three intracellular proteins will result in modulation of the effects caused by this interaction. Thus, inhibition of TRADD binding to MORT-1 may modulate FAS-R-p55 TNF-R intraction. Inhibition of RIP in addition to the above inhibition of TRADD binding to MORT-1 may further modulate FAS-R-p55 TNF-R interaction.
Monoclonal antibodies raised against the xe2x80x98death domainxe2x80x99 of the p55 TNF-R, specifically against the binding site or sites of TRADD and RIP can also be used to inhibit or prevent binding of these proteins and thus cause modulation of the interaction between the FAS-R and the p55 TNF-R.
In a way analogous to that noted above in respect of TNFJNF-R and FAS-ligand/FAS-R, there is also a need to provide a way for modulating the activity of the above noted proteins, i.e. ankyrin, NGF-R and MORT-1, namely, to inhibit their activity when it is associated with detrimental effects, e.g. disease/disorder-related cell cytotoxicity or conformational changes in cell-shape; or to enhance their activity when this is desired, e.g. for directed destruction of diseased cells, etc.
In the co-pending applications, IL 109632, IL 111125, EL 112002 and IL 112692, there are described proteins which are involved in the modulation of the activity of receptors belonging to the TNF/NGF receptor family, these proteins being characterized by being capable of binding/associating with the intracellular domains of one or more of these receptors.
The present invention concerns modulators such as proteins, peptides, antibodies and organic compounds which are capable of interacting/binding with one or more so-called xe2x80x98death domainxe2x80x99 motifs in the intracellular domains of proteins containing such motifs, these proteins being related, e.g. members of the TNF/NGF receptor family or proteins related thereto, e.g. MORT1, or unrelated proteins, e.g. ankyrins. These modulators are characterized by recognizing general structural features common to the xe2x80x98death domainxe2x80x99 motifs of the xe2x80x98death domainxe2x80x99 motif-containing proteins, and by also recognizing specific structural features present in each of the different xe2x80x98death domainxe2x80x99 motifs of these proteins.
Accordingly, it is one aim of the invention to provide modulators, as noted above, capable of binding to or interacting with the xe2x80x98death domainxe2x80x99 motifs of one or more of the xe2x80x98death domainxe2x80x99 motif-containing proteins and thereby modulating the activity of these proteins.
Another aim of the invention is to provide antagonists (e.g. antibodies) to one class of these modulators, namely the naturally-occurring proteins or peptides which bind to xe2x80x98death domainxe2x80x99 motif-containing proteins, and which antagonists may be used to inhibit the signaling process, when desired, when such xe2x80x98death domainxe2x80x99 motif-binding proteins or peptides are positive signal effectors (i.e. induce signaling), or to enhance the signaling process, when desired, when such xe2x80x98death domainxe2x80x99 motif-binding proteins are negative signal effectors (i.e. inhibit signaling).
Yet another aim of the invention is to use such xe2x80x98death domainxe2x80x99 motif-binding proteins or peptides to isolate and characterize additional proteins or factors, which may, for example, be involved further downstream in the signaling process, and/or to isolate and identify other receptors further upstream in the signaling process to which these xe2x80x98death domainxe2x80x99 motif-binding proteins bind, and hence, in whose function they are also involved.
Moreover, it is an aim of the present invention to use the above-mentioned xe2x80x98death domainxe2x80x99 motif-binding proteins as antigens for the preparation of polyclonal and/or monoclonal antibodies thereto. The antibodies. in turn, may be used for the purification of the new xe2x80x98death domainxe2x80x99 motif-binding proteins from different sources, such as cell extracts or transformed cell lines.
Furthermore, these antibodies may be used for diagnostic purposes, e.g. for identifying disorders related to abnormal functioning of cellular effects mediated by the various proteins belonging to the group of xe2x80x98death domainxe2x80x99 motif-containing proteins.
A further aim of the invention is to provide pharmaceutical compositions comprising the above xe2x80x98death domainxe2x80x99 motif-binding modulators (proteins, peptides, organic molecules), and pharmaceutical compositions comprising the xe2x80x98death domainxe2x80x99 motif-binding protein or peptide antagonists, for the treatment or prophylaxis of conditions related to the activity of the xe2x80x98death domainxe2x80x99 motif-containing proteins, for example, such compositions can be used to enhance the TNF or FAS ligand effect or effects mediated by NGF-R, MORT-1, RIP, TRADD and ank-yrin, or to inhibit the TNF or FAS ligand effect or effects mediated by depending on the above noted nature of the xe2x80x98death domainxe2x80x99 motif-binding modulators or antagonists thereof contained in the composition.
A still further aim of the invention is to use the various xe2x80x98death domainxe2x80x99 motifs of the proteins containing them for the design and synthesis of complementary peptides and organic molecules which will be modulators of these proteins.
The present invention is based on the surprising and unexpected finding that there exists a so-called xe2x80x98death domainxe2x80x99 motif in a wide range of proteins some of which are related and others which are not related. For example, this xe2x80x98death domainxe2x80x99 motif has been found in p55 TNF-R, FAS-R, NGF-R, MORTI, RIP and TRADD which are related to each other, as well as in the unrelated protein, ankyrin 1.
As noted above, the xe2x80x98death domainxe2x80x99 motif of the proteins containing this motif is located in the intracellular regulatory domain of these proteins. Hence, the xe2x80x98death domainxe2x80x99 motif appears to be involved in a regulatory function associated with cell viability (cell death) as well as cell shape/conformation, this function being effected at (i.e. in the case of receptors containing this motif) or close to (i.e. in the case of structural intracellular proteins, e.g. ankyrin) the cell surface. Moreover, the observation, in accordance with the present invention, that the xe2x80x98death domainxe2x80x99 motif is conserved amongst a wide range of related and non-related proteins indicates that this motif may have an important regulatory function.
Accordingly, the present invention provides a modulator of regulatory cellular events occurring intracellularly that are mediated by regulatory proteins containing a xe2x80x98death domainxe2x80x99 motif which is a regulatory portion of said proteins, said modulator being capable of interacting with one or more of the xe2x80x98death domainxe2x80x99 motifs contained in said regulatory proteins and affecting the regulatory action of one or more of said regulatory proteins.
In particular, the present invention provides:
(i) a modulator is selected from the group comprising naturally-derived xe2x80x98death domainxe2x80x99 motif-binding proteins and peptides and analogs and derivatives thereof capable of interacting with one or more of said xe2x80x98death domainxe2x80x99 motifs;
(ii) a modulator is selected from the group of synthetically produced complementary peptides, synthesized by using as substrates the xe2x80x98death domainxe2x80x99 motif sequences of said regulatory proteins containing xe2x80x98death domainxe2x80x99 motifs, said complementary peptides being capable of interacting with one or more of said xe2x80x98death domainxe2x80x99 motifs.
(iii) a modulator is selected from the group comprising antibodies or active fragments thereof capable of interacting with one or more of said xe2x80x98death domainxe2x80x99 motifs.
(iv) a modulator is selected from the group of organic compounds capable of interacting with one or more of said xe2x80x98death domainxe2x80x99 motifs, said organic compounds being derived from known compounds and selected by using said xe2x80x98death domainxe2x80x99 motifs as a substrate in a binding assay, or being synthesized using said xe2x80x98death domainxe2x80x99 motifs as a substrate for designing and synthesizing said organic compounds.
(v) a modulator is selected from the group of peptides or polypeptides derived from naturally occurring xe2x80x98death domainxe2x80x99 motif sequences, said peptides or polypeptides being capable of interacting with one or more of said xe2x80x98death domainxe2x80x99 motifs, and analogs and derivatives of said peptides or polypeptides capable of interacting with one or more of said xe2x80x98death domainxe2x80x99 motifs.
(vi) a modulator of any one of (i)-(v) wherein said modulator is further characterized by being capable of recognizing the general xe2x80x98death domainxe2x80x99 motif sequence features common to the xe2x80x98death domainxe2x80x99 motifs of xe2x80x98death domainxe2x80x99 motif containing proteins, and being capable of recognizing one or more of the specific xe2x80x98death domainxe2x80x99 motifs of said proteins, said specific sequence features being specific to each xe2x80x98death domainxe2x80x99 motif sequence of each of said proteins.
(vii) a modulator of any one of (i)-(vi) wherein said modulator is capable of interacting with one or more of the xe2x80x98death domainxe2x80x99 motifs contained within the proteins belonging to the group comprising p55 TNF-R, FAS-R, NGF-R, MORT-1, RIP, TRADD and ankyrin 1.
(viii) a modulator of (vii) wherein said modulator is further characterized by being capable of interacting with common sequence features of the xe2x80x98death domainxe2x80x99 motifs of said group of proteins, said common sequence features comprising the group of common amino acid residues W (tryptophan), L (leucine), I (isoleucine), A (alanine), D (aspartic acid), E (glutamic acid), T (threonine), R (arginine) and Y (tyrosine) at the location within said xe2x80x98death domainxe2x80x99 motifs shown in FIG. 1.
The present invention also provides a DNA sequence encoding a modulator being a protein, peptide or polypeptide or an analog of any one of (i), (ii) and (vii).
An embodiment of the DNA sequence of the invention is a DNA sequence encoding a naturally derived protein or peptide selected from the group consisting of:
(a) a cDNA sequence derived from the coding region of a native xe2x80x98death domainxe2x80x99 motif-binding protein or peptide,
(b) DNA sequences capable of hybridization to a sequence of (a) under moderately stringent conditions and which encode a biologically active xe2x80x98death domainxe2x80x99 motif-binding protein or peptide; and
(c) DNA sequences which are degenerate as a result of the genetic code to the DNA sequences defined in (a) and (b) and which encode a biologically active xe2x80x98death domainxe2x80x99 motif-binding protein or peptide.
Other embodiments of the DNA sequence of the invention are:
(i) DNA sequence encoding a xe2x80x98death domainxe2x80x99 motif-binding protein or peptide capable of binding to the xe2x80x98death domainxe2x80x99 motif of one or more of the proteins of the group comprising p55 TNF-R, FAS-R, NGF-R, MORT-1 and anlvrin 1.
(ii) DNA sequence encoding a peptide or polypeptide derived from the naturally occurring xe2x80x98death domainxe2x80x99 motif sequence of the xe2x80x98death domainxe2x80x99 motif-containing proteins.
(iii) DNA sequence encoding a peptide or polypeptide derived from the xe2x80x98death domainxe2x80x99 motif sequence of any one of the proteins of the group comprising p55 TNF-R, FAS-R, NGF-R, MORT-1, RIP, TRADD and ankyrin 1.
Furthermore, there is also provided:
(a) a protein, peptide or polypeptide and analogs of any one thereof encoded by a DNA sequence of the invention. said protein, peptide, polypeptide and analogs being capable of binding to or interacting with one or more of the xe2x80x98death domainxe2x80x99 motifs of one or more xe2x80x98death domainxe2x80x99 motif containing proteins.
(b) a vector comprising a DNA sequence of the invention.
(c) a vector of (b) capable of being expressed in a eukaryotic host cell.
(d) a vector of (b) capable of being expressed in a prokaryotic host cell.
(e) transformed eukaryotic or prokaryotic host cells containing a vector of (b), (c) or (d)
(f) a method for producing the protein, peptide, polypeptide or analogs of (a) comprising growing the transformed host cells of (e) under conditions suitable for the expression of said protein, peptide, polypeptide or analogs, effecting post-translational modifications of said protein, peptide, polypeptide or analogs as necessary for obtention thereof and extracting said expressed protein, peptide, polypeptide or analogs from the culture medium of said transformed cells or from cell extracts of said transformed cells.
(g) antibodies or active fragments or derivatives thereof, specific for the protein, peptide, polypeptide or analogs of (a).
The present invention also provides a method for the modulation of the TNF or FAS-R ligand effect on cells mediated by p55 TNF-R and FAS-R, or the functions mediated in cells by NGF-R, MORT-1, RIP, TRADD, ankyrin 1 or by other proteins containing a xe2x80x98death domainxe2x80x99 motif, comprising treating said cells with one or more proteins, peptides, polypeptides or analogs selected from the group consisting of the proteins, peptides, polypeptides or analogs of the invention (see (a) above), all being capable of binding to or interacting with the xe2x80x98death domainxe2x80x99 motif and modulating the activity of said xe2x80x98death domainxe2x80x99 motif-containing proteins, wherein said treating of said cells comprises introducing into said cells said one or more proteins, peptides, polypeptides or analogs in a form suitable for intracellular introduction thereof, or introducing into said cells a DNA sequence encoding said one or more proteins, peptides, polypeptides or analogs in the form of a suitable vector carrying said sequence, said vector being capable of effecting the insertion of said sequence into said cells in a way that said sequence is expressed in said cells.
An embodiment of the above method is a method wherein said treating of said cells is by transfection of said cells with a recombinant animal virus vector comprising the steps of:
(a) constructing a recombinant animal virus vector carrying a sequence encoding a viral surface protein (ligand) that is capable of binding to a specific cell surface receptor on the surface of said cell to be treated and a second sequence encoding a protein selected from the proteins, peptides, polypeptides and analogs of the invention, said protein, peptide, polypeptide or analogs, when expressed in said cells being capable of modulating the activity of said xe2x80x98death domainxe2x80x99 motif-containing protein; and
(b) infecting said cells with said vector of (a).
Another method of the invention is a method for modulating the TNF or FAS-R ligand effect on cells mediated by p55 TNF-R and FAS-R, or the functions mediated in cells by NGF-R, MORT-1,RIP, TRADD, ankyrin 1 or by other proteins containing a xe2x80x98death domainxe2x80x99 motif, comprising treating said cells with antibodies or active fragments or derivatives thereof, of the invention (see (g) above), said treating being by application of a suitable composition containing said antibodies, active fragments or derivatives thereof to said cells, said composition being formulated for intracellular application.
Yet another method of the invention is a method for modulating the TNF or FAS-R ligand effect on cells mediated by p55 TNF-R and FAS-R, or the functions mediated in cells by NGF-R, MORT-1, RIP, TRADD, ankyrin 1 or by other proteins containing a xe2x80x98death domainxe2x80x99 motif, comprising treating said cells with an oligonucleotide sequence selected from a sequence encoding an antisense sequence of at least part of the sequence of the invention as noted above, said oligonucleotide sequence being capable of blocking the expression of at least one of the xe2x80x98death domainxe2x80x99 motif-binding proteins or peptides.
An embodiment of the above method is a method wherein said oligonucleotide sequence is introduced to said cells via a virus vector as noted above wherein said second sequence of said virus encodes said oligonucleotide sequence.
Other methods of the invention are:
(i) a method for treating tumor cells or HIV-infected cells or other diseased cells, comprising:
(a) constructing a recombinant animal virus vector carrying a sequence encoding a viral surface protein that is capable of binding to a specific tumor cell surface receptor or HIV-infected cell surface receptor or receptor carried by other diseased cells and a sequence encoding a protein selected from the proteins, peptides, polypeptides and analogs of the invention, said protein, peptide, polypeptide or analogs when expressed in said tumor, HIV-infected, or other diseased cell being capable of killing said cell; and
(b) infecting said tumor or HIV-infected cells or other diseased cells with said vector of (a).
(ii) a method for modulating the TNF or FAS-R ligand effect on cells mediaed by p55 TNF-R and FAS-R, or the fuinctions mediated in cells by NGF-R, MORT-1, RIP, TRADD, ankyrin 1 or by other proteins containing a xe2x80x98death domainxe2x80x99 motif, comprising applying the ribozyme procedure in which a vector encoding a ribozyme sequence capable of interacting with a cellular mRNA sequence encoding a protein or peptide of the invention, is introduced into said cells in a form that permits expression of said ribozyne sequence in said cells, and wherein when said ribozyme sequence is expressed in said cells it interacts with said cellular mRNA sequence and cleaves said mRNA sequence resulting in the inhibition of expression of said protein or peptide in said cells.
(iii) a method for isolating and identifying proteins, peptides, factors or receptors capable of binding to the xe2x80x98death domainxe2x80x99 motif-binding proteins or peptides of the invention, comprising applying the procedure of affinity chromatography in which said protein or peptide of the invention is attached to the affinity chromatography matrix, said attached protein is brought into contact with a cell extract and proteins, factors or receptors from cell extract which bound to said attached protein are then eluted, isolated analyzed.
(iv) a method for isolating and identifying proteins, capable of binding to the xe2x80x98death domainxe2x80x99 motif-binding proteins or peptides of the invention, comprising applying the yeast two-hybrid procedure in which a sequence encoding said xe2x80x98death domainxe2x80x99 motif-binding protein is carried by one hybrid vector and sequence from a cDNA or genomic DNA library are carried by the second hybrid vector, the vectors then being used to transform yeast host cells and the positive transformed cells being isolated, followed by extraction of the said second hybrid vector to obtain a sequence encoding a protein which binds to said xe2x80x98death domainxe2x80x99 motif-binding protein.
The present invention also provides a pharmaceutical composition for the modulation of the TNF- or FAS-R ligand- effect on cells mediated by p55 TNF-R and FAS-R, or the fuinctions mediated in cells by NGF-R, MORT-1, RIP, TRADD, ankyrin 1 or by other proteins containing a xe2x80x98death domainxe2x80x99 motif comprising, as active, ingredient a modulator of the invention.
Embodiments of the pharmaceutical compositions of the invention include:
(i) a pharmaceutical composition for modulating the TNF- or FAS-R ligand-effect on cells mediated by p55 TNF-R and FAS-R, or the functions mediated in cells by NGF-R, MORT-1, RIP, TRADD, ankyrin 1 or by other proteins containing a xe2x80x98death domainxe2x80x99 motif, comprising, as active ingredient, a recombinant animal virus vector encoding a protein capable of binding a cell surface receptor and encoding a protein or peptide or analogs thereof of the invention.
(ii) a pharmaceutical composition for modulating the TNF or FAS-R ligand effect on cells mediated by p55 TNF-R and FAS-R, or the functions mediated in cells by NGF-R, MORT-1, RIP, TRADD, ankyrin 1 or by other proteins containing a xe2x80x98death domainxe2x80x99 motif, comprising as active ingredient, an oligonucleotide sequence encoding an anti-sense sequence of the sequence of the invention.
A still further method of the invention is a method for isolating and identifying a protein capable of binding to the xe2x80x98death domainxe2x80x99 motifs of xe2x80x98death domainxe2x80x99 motif-containing proteins comprising applying the procedure of non-stringent southern hybridization followed by PCR cloning, in which a sequence or parts thereof of the invention is used as a probe to bind sequences from a cDNA or genomic DNA library, having at least partial homology thereto, said bound sequences then amplified and cloned by the PCR procedure to yield clones encoding proteins having at least partial; homology to said sequences of the invention.
In addition, the present invention also provides a method for designing drugs that are capable of modulating the activity of xe2x80x98death domainxe2x80x99 motif-containing proteins, comprising the procedures described herein in Examples 3 and 4.
Other aspects and embodiments of the present invention are also provided as arising from the following detailed description of the invention.
It should be noted that, where used throughout, the following terms xe2x80x9cModulation/Mediation of the TNF or FAS-R ligand effect on cells mediated by p55 TNF-R and FAS-R, or the flunctions mediated in cells by NGF-R, MORT1, RIP, TRADD, ankyrin 1 or by other proteins containing a xe2x80x98death domainxe2x80x99 motif are understood to encompass in vitro as well as in vivo treatment.
Moreover, where used throughout, the antibodies of the invention and the methods using these antibodies, include so-called xe2x80x9chumanizedxe2x80x9d antibodies or the use thereof.